Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector

Lussier, François-Xavier; Chambenoit, Olivier; Côté, Amélie; Hupé, Jean-François; Denis, François; Juteau, Pierre; Beaudet, Réjean; Shareck, François

doi:10.1007/s10295-010-0915-2

Cited by 21 publications

(13 citation statements)

References 43 publications

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“…To overcome these restrictions, other screening hosts have been implemented in function-based metagenomic screening approaches in order to increase the detection frequency of active clones. First attempts using additional screening hosts involved Streptomyces and Pseudomonas species (Courtois et al, 2003 ; Martinez et al, 2004 ; Lussier et al, 2011 ). By the use of broad-host range vectors, the expression could recently be extended to other members of the Proteobacteria , i.e., the same taxon as E. coli (Aakvik et al, 2009 ; Craig et al, 2010 ; Kakirde et al, 2011 ), and to eukaryotic expression hosts (Damon et al, 2011 ; Kellner et al, 2011 ; Parachin and Gorwa-Grauslund, 2011 ).…”

Section: Discussionmentioning

confidence: 99%

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

et al. 2015

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Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

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Section: Discussionmentioning

confidence: 99%

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

et al. 2015

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“…One of the most recent studies proves its effectiveness using six different proteobacteria as hosts for the same metagenomic cosmid library, recovering different positive clones in each host [36]. More recently, Lussier and collaborators [37] developed a cosmid vector with two improvements: multi-host expression ( E. coli and Streptomyces lividans ) and transcription directed by T7 RNA polymerase, which has high activity, generates very long mRNAs and is very poorly terminated by unrelated transcription terminators, potentially enhancing the expression of foreign genes in large insert libraries, in this case lipase/esterase genes. A special fosmid vector has even been developed for the expression of metagenomic libraries from thermophiles, which allows the library to be constructed in E. coli and subsequently transferred to Thermus thermophilus for expression and screening [38].…”

Section: Metagenomics In the Discovery Of New Lipases And Esterasesmentioning

confidence: 99%

New Extremophilic Lipases and Esterases from Metagenomics

López-López¹,

Cerdán

Siso

2014

CPPS

155

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Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications.

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“…9 Nevertheless, previous attempts at functional metagenomic screening in Streptomyces , which have been conducted using exclusively Streptomyces lividans as a host, have largely been unsuccessful at identifying secondary metabolites. 10–14 …”

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confidence: 99%

Natural Product Discovery through Improved Functional Metagenomics in Streptomyces

et al. 2016

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Because the majority of environmental bacteria are not easily culturable, access to many bacterially encoded secondary metabolites will be dependent on the development of improved functional metagenomic screening methods. In this study, we examined a collection of diverse Streptomyces species for the best innate ability to heterologously express biosynthetic gene clusters. We then optimized methods for constructing high quality meta-genomic cosmid libraries in the best Streptomyces host. An initial screen of a 1.5 million-membered metagenomic library constructed in Streptomyces albus, the species that exhibited the highest propensity for heterologous expression of gene clusters, led to the identification of the novel natural product metatricycloene (1). Metatricy-cloene is a tricyclic polyene encoded by a reductive, iterative polyketide-like gene cluster. Related gene clusters found in sequenced genomes appear to encode a largely unexplored collection of structurally diverse, polyene-based metabolites.

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Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector

Cited by 21 publications

References 43 publications

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

New Extremophilic Lipases and Esterases from Metagenomics

Natural Product Discovery through Improved Functional Metagenomics in Streptomyces

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